RESUMEN
During development, endothelial tip cells (ETCs) located at the leading edge of growing vascular plexus guide angiogenic sprouts to target vessels, and thus, ETC pathfinding is fundamental for vascular pattern formation in organs, including the brain. However, mechanisms of ETC pathfinding remain largely unknown. Here, we report that Piezo1-mediated Ca2+ activities at primary branches of ETCs regulate branch dynamics to accomplish ETC pathfinding during zebrafish brain vascular development. ETC branches display spontaneous local Ca2+ transients, and high- and low-frequency Ca2+ transients cause branch retraction through calpain and branch extension through nitric oxide synthase, respectively. These Ca2+ transients are mainly mediated by Ca2+-permeable Piezo1 channels, which can be activated by mechanical force, and mutating piezo1 largely impairs ETC pathfinding and brain vascular patterning. These findings reveal that Piezo1 and downstream Ca2+ signaling act as molecular bases for ETC pathfinding and highlight a novel function of Piezo1 and Ca2+ in vascular development.
Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Encéfalo/irrigación sanguínea , Calcio/metabolismo , Células Endoteliales/metabolismo , Canales Iónicos/genética , Neovascularización Fisiológica/genética , Proteínas de Pez Cebra/genética , Animales , Encéfalo/crecimiento & desarrollo , Señalización del Calcio , Calpaína/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular , Mutación , Óxido Nítrico Sintasa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistemas CRISPR-Cas/genética , Marcación de Gen/métodos , Recombinación Homóloga/genética , Intrones/genética , Proteínas de Pez Cebra/genética , Alelos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Expresión Génica , Técnicas de Sustitución del Gen/métodos , Técnicas de Inactivación de Genes/métodos , Ingeniería Genética , Genotipo , Proteínas Fluorescentes Verdes/genética , Pez CebraRESUMEN
Vascular integrity helps maintain brain microenvironment homeostasis, which is critical for the normal development and function of the central nervous system. It is known that neural cells can regulate brain vascular integrity. However, due to the high complexity of neurovascular interactions involved, understanding of the neural regulation of brain vascular integrity is still rudimentary. Using intact zebrafish larvae and cultured rodent brain cells, we find that neurons transfer miR-132, a highly conserved and neuron-enriched microRNA, via secreting exosomes to endothelial cells (ECs) to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase (eef2k). Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity. Our study indicates that miR-132 acts as an intercellular signal mediating neural regulation of the brain vascular integrity and suggests that the neuronal exosome is a novel avenue for neurovascular communication.
